Professor Hubert Hondermarck

The prolonged war in Ukraine is having a strong impact on all sectors of the Ukrainian society, including biomedical research. Although the material and psychological conditions are challenging, the country and its researchers are courageously managing to continue their activities. This perspective paper describes the multiple challenges faced by Ukrainian biomedical researchers during wartime and outlines strategies to support and enhance collaboration with the global scientific community. Ukraine has a rich scientific history and modern expertise in biomedical research, and developing more international collaborations with Ukraine can have mutual benefits for all involved parties.

Nerve dependence in regeneration has been established more than 200¿years ago but the mechanisms by which nerves are necessary to regeneration remain to be fully elucidated. Aside from their direct impact in stimulating cellular growth, nerves also have a role on the establishment of body polarities (antero-posterior and dorso-ventral patterns) and this has been particularly well studied in nereid annelid worms. Nereids can regenerate appendages (parapodia) and the tail (body segments). In both parapodia and tail regeneration, the presence of the nerve cord is necessary to the establishment of body polarities. In this review, we will detail the experimental procedures which have been conducted in nereids to elucidate the role of the nerve cord in the establishment of the antero-posterior and dorso-ventral polarities. Most of the studies reported here were published several decades ago and based on anatomical and histological analyses; this review should constitute a knowledgebase and an inspiration for needed modern-time explorations at the molecular levels to elucidate the impact of the nervous system in the acquisition of body polarities.

Active crosstalk between the nervous system and breast cancer cells has been experimentally demonstrated in vitro and in animal models. However, low frequencies of peripheral nerve presence in human breast cancers reported in previous studies (~30% of cases) potentially negate a major role of the nervous system in breast cancer development and progression. This study aimed to clarify the incidence of nerves within human breast cancers and to delineate associations with clinicopathological features. Immunohistochemical staining was conducted in formalin-fixed paraffin-embedded breast cancer tissue sections using antibodies against the pan-neuronal markers protein gene product 9.5 and growth-associated protein 43, and the sympathetic nerve-specific marker tyrosine hydroxylase. Nerve trunks and isolated nerve fibers were quantitated. The chi-squared test was used to determine the associations between nerve counts and clinicopathological parameters. The log-rank test was used to compare differences in patient progression-free survival (PFS) and overall survival (OS). The overall frequency of peripheral nerves in breast cancers was 85%, a markedly higher proportion than reported previously. Of note, most nerves present in breast cancers were of the sympathetic origin. While high density of nerve trunks or isolated nerve fibers was associated with poor PFS and OS of patients, high nerve trunk density appeared also to predict poor patient PFS independently of lymph node metastasis. Innervation of breast cancers is a common event correlated with poor patient outcomes. These findings support the notion that the nervous system plays an active role in breast cancer pathogenesis.

Previous reports have shown that quantification of high tumour grade is of prognostic significance for patients with prostate cancer. In particular, percent Gleason pattern 4 (GP4) has been shown to predict outcome in several studies, although conflicting results have also been reported. A major issue with these studies is that they rely on surrogate markers of outcome rather than patient survival. We have investigated the prognostic predictive value of quantifying GP4 in a series of prostatic biopsies containing Gleason score 3+4=7 and 4+3=7 tumours. It was found that the length of GP4 tumour determined from the measurement of all biopsy cores from a single patient, percent GP4 present and absolute GP4 were all significantly associated with distant progression of tumour, all-cause mortality and cancer-specific mortality over a 10-year follow-up period. Assessment of the relative prognostic significance showed that these parameters outperformed division of cases according to Gleason score (3+4=7 versus 4+3=7). International Society of Urological Pathology (ISUP) Grade Groups currently divide these tumours, according to Gleason grading guidelines, into grade 2 (3+4=7) and grade 3 (4+3=7). Our results indicate that this simple classification results in the loss of important prognostic information. In view of this we would recommend that ISUP Grade Groups 2 and 3 be amalgamated as grade 2 tumour with the percentage of GP4 carcinoma being appended to the final grade, e.g., 3+4=7 carcinoma with 40% pattern 4 tumour would be classified as ISUP Grade Group 2 (40%).

Non-invasive procedures are needed for prostate cancer management, and urine represents a potential source of new biomarkers with translational value. Recent evidence has shown that the growth of new nerves in the tumor microenvironment is essential to prostate cancer progression. Neurotrophic growth factors are expressed by prostate cancer cells and contribute to prostate tumor innervation, but their presence in urine is unclear. In the present study, we have assayed the concentration of neurotrophic factors in the urine of prostate cancer patients. Urine was collected from a prospective cohort of 45 men with prostate cancer versus 30 men without cancer and enzyme-linked immunosorbent assay was used to quantify nerve growth factor (NGF) and its precursor proNGF, brain-derived neurotrophic factor (BDNF) and proBDNF, neurotrophin-3, neurotrophin-4/5, and glia-derived neurotrophic growth factor. The results show that neurotrophic factors are detectable in various concentrations in both cancer and healthy urine, but no significant difference was found. Also, no association was observed between neurotrophic factor concentrations and prostate cancer grade. This study is the first quantification of neurotrophins in urine, and although no significant differences were observed between prostate cancer patients versus those without prostate cancer, or between prostate cancers of various grades, the potential value of neurotrophins for prostate cancer diagnosis and prognosis warrants further investigations in larger patient cohorts.

Nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA) and the common neurotrophin receptor (NGFR/p75NTR), are increasingly implicated in cancer progression, but their clinicopathological significance in oesophageal cancer is unclear. In this study, the expression of NGF, NTRK1 and NGFR were analysed by immunohistochemistry in a cohort of 303 oesophageal cancers versus 137 normal adjacent oesophageal tissues. Immunostaining was digitally quantified and compared to clinicopathological parameters. NGF and NGFR staining were found in epithelial cells and at similar levels between oesophageal cancers and normal oesophageal tissue. NGFR staining was slightly increased with grade (p=0.0389). Interestingly, NTRK1 staining was markedly higher in oesophageal squamous cell carcinoma (OR 2.31, 95%CI 1.13¿4.38, p<0.0001) and significantly lower in adenocarcinoma (OR 0.50, 95%CI 0.44¿0.63, p<0.0001) compared to normal oesophageal tissue. In addition, NTRK1 staining was decreased in grade 2 and grade 3 (OR 0.51, 95%CI 0.21¿1.40, p<0.0001) compared to grade 1, suggesting a preferential involvement of this receptor in the more differentiated forms of oesophageal carcinomas. Together, these data point to NTRK1 as a biomarker and a candidate therapeutic target in oesophageal squamous cell carcinoma.

Although the role of nerves in stimulating cellular growth and dissemination has long been described in tissue regeneration studies, until recently a similar trophic role of nerves in disease was not well recognized. However, recent studies in oncology have demonstrated that the growth and dissemination of cancers also requires the infiltration of nerves in the tumor microenvironment. Nerves generate various neurosignaling pathways, which orchestrate cancer initiation, progression, and metastases. Similarly, nerves are increasingly implicated for their regulatory functions in immunity and inflammation. This orchestrator role of nerves in cellular and molecular interactions during regeneration, cancer, immunity, and inflammation offers new possibilities for targeting or enhancing neurosignaling in human health and diseases.

Nerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell¿released NGF.

Aims: Perineural invasion (PNI) by prostatic adenocarcinoma is debated as a prognostic parameter. This study investigates the prognostic predictive value of PNI in a series of patients with locally advanced prostate cancer treated with radiotherapy and androgen deprivation using 10¿years outcome data from the TROG 03.04 RADAR trial. Methods: Diagnostic prostate biopsies from 976 patients were reviewed and the presence of PNI noted. Patients were followed for 10¿years according to the trial protocol or until death. The primary endpoint for the study was time to bone metastasis. Secondary endpoints included time to soft tissue metastasis, transition to castration resistance, prostate cancer-specific mortality and all-cause mortality. Results: PNI was detected in 449 cases (46%), with 234 cases (24%) having PNI in more than one core. The presence of PNI was significantly associated with higher ISUP grade, clinical T staging category, National Comprehensive Cancer Network risk group, and percent positive biopsy cores. The cumulative probability of bone metastases according to PNI status was significant over the 10¿years follow-up interval of the study (log-rank test P¿<¿0.0001). PNI was associated with all endpoints on univariable analysis. After adjusting for baseline clinicopathological and treatment factors, bone metastasis was the only endpoint in which PNI retained its prognostic significance (hazard ratio 1.42, 95% confidence interval 1.05¿1.92, P¿=¿0.021). Conclusions: The association between PNI and the development of bone metastases supports the inclusion of this parameter as a component of the routine histology report. Further this association suggests that evaluation of PNI may assist in selecting those patients who should be monitored more closely during follow-up.

The tyrosine kinase receptor A (NTRK1/TrkA) is increasingly regarded as a therapeutic target in oncology. In breast cancer, TrkA contributes to metastasis but the clinicopathological significance remains unclear. In this study, TrkA expression was assessed via immunohistochemistry of 158 invasive ductal carcinomas (IDC), 158 invasive lobular carcinomas (ILC) and 50 ductal carcinomas in situ (DCIS). TrkA was expressed in cancer epithelial and myoepithelial cells, with higher levels of TrkA positively associated with IDC (39% of cases) (p < 0.0001). Interestingly, TrkA was significantly increased in tumours expressing the human epidermal growth factor receptor-2 (HER2), with expression in 49% of HER2-positive compared to 25% of HER2-negative tumours (p = 0.0027). A panel of breast cancer cells were used to confirm TrkA protein expression, demonstrating higher levels of TrkA (total and phosphorylated) in HER2-positive cell lines. Functional investigations using four different HER2-positive breast cancer cell lines indicated that the Trk tyrosine kinase inhibitor GNF-5837 reduced cell viability, through decreased phospho-TrkA (Tyr490) and downstream AKT (Ser473) activation, but did not display synergy with Herceptin. Overall, these data highlight a relationship between the tyrosine kinase receptors TrkA and HER2 and suggest the potential of TrkA as a novel or adjunct target for HER2-positive breast tumours.

Prostate cancer progression has been shown to be dependent on the development of autonomic nerves into the tumour microenvironment. Sympathetic nerves activate adrenergic neurosignalling that is necessary in early stages of tumour progression and for initiating an angiogenic switch, whereas parasympathetic nerves activate cholinergic neurosignalling resulting in tumour dissemination and metastasis. The innervation of prostate cancer seems to be initiated by neurotrophic growth factors, such as the precursor to nerve growth factor secreted by tumour cells, and the contribution of brain-derived neural progenitor cells has also been reported. Current experimental, epidemiological and clinical evidence shows the stimulatory effect of tumour innervation and neurosignalling in prostate cancer. Using nerves and neurosignalling could have value in the management of prostate cancer by predicting aggressive disease, treating localized disease through denervation and relieving cancer-associated pain in bone metastases.

Increasing knowledge of how peptides bind saccharides, and of how saccharides bind peptides, is starting to revolutionize understanding of cell-extracellular matrix relationships. Here, a historical perspective is taken of the relationship between heparan sulfate glycosaminoglycans and how they interact with peptide growth factors in order to both drive and modulate signaling through the appropriate cognate receptors. Such knowledge is guiding the preparation of targeted sugar mimetics that will impact the treatment of many different kinds of diseases, including cancer.

Nerves are emerging regulators of cancer progression. Cancer cells induce the outgrowth of nerves in the tumor microenvironment through the release of neu-rotrophic factors, and in return nerves liberate neurotransmitters that activate cancer growth and dissemination. Although sympathetic nerves drive tumor angiogenesis via the liberation of noradrena-line, sensory and parasympathetic nerves stimulate cancer stem cells. Interestingly, recent evidence indicates that parasympathetic nerves can eventually inhibit tumor progression, suggesting a yin¿yang type of regulation of cancer by nerves. From a broader perspective, the question of a higher level of control of cancer development by the central nervous system should be raised. Significance: Nerves are emerging regulators of cancer initiation, progression, and metastasis. Here, we review the evidence to date and explore the basic and clinical ramifications of these findings.

Nerve outgrowth in the tumor microenvironment (tumor neurogenesis) has recently been shown to be essential for cancer progression and the concept of nerve dependence is emerging in oncology. Neurotrophins such as nerve growth factor (NGF) have long been identified as drivers of neurogenesis during development and regeneration, but intriguingly they were also known to be expressed in human tumors where they can stimulate cancer cell growth. Recent findings have unraveled that NGF released by cancer cells is also a driver of tumor neurogenesis, via the stimulation of NGF receptors on nerve endings. In return, nerves infiltrated in the tumor microenvironment secrete neurotransmitters, which can stimulate both the growth of tumor cells and angiogenesis. This neurotrophic role of NGF in cancer is likely to be relevant to a large variety of human malignancies, as well as other neurotrophins, and may have ramifications in cancer pain. Therefore, pharmacological interventions against neurotrophin signaling have the potential not only to target cancer cells directly, but also to inhibit neurogenesis and its stimulatory impact on cancer progression and pain.

Neurotrophin receptors are emerging targets in oncology, but their clinicopathologic significance in thyroid cancer is unclear. In this study, the neurotrophin tyrosine receptor kinase TrkA (also called NTRK1), the common neurotrophin receptor p75NTR, and the proneurotrophin receptor sortilin were analyzed with immunohistochemistry in a cohort of thyroid cancers (n = 128) and compared with adenomas and normal thyroid tissues (n = 62). TrkA was detected in 20% of thyroid cancers, compared with none of the benign samples (P = 0.0007). TrkA expression was independent of histologic subtypes but associated with lymph node metastasis (P = 0.0148), suggesting the involvement of TrkA in tumor invasiveness. Nerves in the tumor microenvironment were positive for TrkA. p75NTR was overexpressed in anaplastic thyroid cancers compared with papillary and follicular subtypes (P < 0.0001). Sortilin was overexpressed in thyroid cancers compared with benign thyroid tissues (P < 0.0001). Neurotrophin receptor expression was confirmed in a panel of thyroid cancer cell lines at the mRNA and protein levels. Functional investigations using the anaplastic thyroid cancer cell line CAL-62 found that siRNA against TrkA, p75NTR, and sortilin decreased cell survival and cell migration through decreased SRC and ERK activation. Together, these data reveal TrkA, p75NTR, and sortilin as potential therapeutic targets in thyroid cancer.

The central characteristic of uterine fibroids is excessive deposition of extracellular matrix (ECM), which contributes to fibroid growth and bulk-type symptoms. Despite this, very little is known about patterns of ECM protein expression in fibroids and whether these are influenced by the most common genetic anomalies, which relate to MED12. We performed extensive genetic and proteomic analyses of clinically annotated fibroids and adjacent normal myometrium to identify the composition and expression patterns of ECM proteins in MED12 mutation-positive and mutation-negative uterine fibroids. Genetic sequencing of tissue samples revealed MED12 alterations in 39 of 65 fibroids (60%) from 14 patients. Using isobaric tagged-based quantitative mass spectrometry on three selected patients (n = 9 fibroids), we observed a common set of upregulated (.1.5-fold) and downregulated (,0.66-fold) proteins in small, medium, and large fibroid samples of annotated MED12 status. These two sets of upregulated and downregulated proteins were the same in all patients, regardless of variations in fibroid size and MED12 status. We then focused on one of the significant upregulated ECM proteins and confirmed the differential expression of periostin using western blotting and immunohistochemical analysis. Our study defined the proteome of uterine fibroids and identified that increased ECM protein expression, in particular periostin, is a hallmark of uterine fibroids regardless of MED12 mutation status. This study sets the foundation for further investigations to analyze the mechanisms regulating ECM overexpression and the functional role of upregulated ECM proteins in leiomyogenesis.

Nerve dependence has long been described in animal regeneration, where the outgrowth of axons is necessary to the reconstitution of lost body parts and tissue remodeling in various species. Recent discoveries have demonstrated that denervation can suppress tumor growth and metastasis, pointing to nerve dependence in cancer. Regeneration and cancer share similarities in regard to the stimulatory role of nerves, and there are indications that the stem cell compartment is a preferred target of innervation. Thus, the neurobiology of cancer is an emerging discipline that opens new perspectives in oncology.

Nerves are emerging as drivers of tumorigenesis, as demonstrated in the mouse where denervation suppresses prostate cancer; however, clinical evidence is needed. Patients with spinal cord injuries (SCIs) resulting in functional denervation of the prostate have a lower incidence of prostate cancer. This may constitute a clinical evidence for nerve dependence in human prostate tumorigenesis.

Globally, ~1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.

Aim: To develop matrix metalloproteinase-responsive gelatin-albumin hybrid nanoparticles encapsulating a selective tropomyosin receptor kinase A (TrkA) inhibitor GNF-5837 (Gel-Alb-GNF HNPs) and to demonstrate their anticancer effects in breast cancer. Methods: Gel-Alb-GNF HNPs were prepared using a pH-controlled complexation process from cationic gelatin, dextran sulfate and albumin-bound GNF-5837. The anticancer activities of Gel-Alb-GNF HNPs were tested in a panel of subtype-specific breast cancer cell lines. Results: Gel-Alb-GNF HNPs (~130 nm) displayed excellent stability and matrix metalloproteinase-triggered drug release. Compared with GNF-5837 alone, Gel-Alb-GNF HNPs not only significantly enhanced the antiproliferative and anti-invasive effects but also restored the apoptosis of cancer cells. Conclusion: Gel-Alb-GNF HNPs may be adaptable for stand-alone therapies or used in combination with traditional chemotherapies for breast cancer treatment.

The precursor for nerve growth factor (proNGF) is expressed in some cancers but its clinicopathological significance is unclear. The present study aimed to define the clinicopathological significance of proNGF in thyroid cancer. ProNGF expression was analysed by immunohistochemistry in two cohorts of cancer versus benign tumors (adenoma) and normal thyroid tissues. In the first cohort (40 thyroid cancers, 40 thyroid adenomas and 80 normal thyroid tissues), proNGF was found overexpressed in cancers compared to adenomas and normal samples (p<0.0001). The area under the receiver-operating characteristic (ROC) curve was 0.84 (95% CI 0.75-0.93, p<0.0001) for cancers versus adenomas, and 0.99 (95% CI 0.98-1.00, p<0.0001) for cancers versus normal tissues. ProNGF overexpression was confirmed in a second cohort (127 cancers of various histological types and 55 normal thyroid tissues) and using a different antibody (p<0.0001). ProNGF staining intensity was highest in papillary carcinomas compared to other histological types (p<0.0001) and there was no significant association with age, gender, tumor size, stage and lymph node status. In conclusion, proNGF is increased in thyroid cancer and should be considered as a new potential diagnostic biomarker.

The use of nanotechnology has great potentials to revolutionize the future cancer diagnosis and therapy. In this context, various nanoparticles (NPs) have been developed for targeted delivery of diagnostic/therapeutic agents to the tumor sites, which thus result in greater efficacy and much less side effects. The targeting property of NPs is often achieved by functionalizing their surface with tumor-specific ligands, such as antibodies, peptides, small molecules and oligonucleotides. In this review, we will discuss recent progress in the multifunctional design of antibody-targeted NPs with a special focus on liposomal, polymeric and protein-based delivery systems.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum-and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.

Brain metastases are a devastating consequence of cancer and currently there are no specific biomarkers or therapeutic targets for risk prediction, diagnosis, and treatment. Here the proteome of the brain metastatic breast cancer cell line 231-BR has been compared with that of the parental cell line MDA-MB-231, which is also metastatic but has no organ selectivity. Using SILAC and nanoLC-MS/MS, 1957 proteins were identified in reciprocal labeling experiments and 1584 were quantified in the two cell lines. A total of 152 proteins were confidently determined to be up- or down-regulated by more than twofold in 231-BR. Of note, 112/152 proteins were decreased as compared with only 40/152 that were increased, suggesting that down-regulation of specific proteins is an important part of the mechanism underlying the ability of breast cancer cells to metastasize to the brain. When matched against transcriptomic data, 43% of individual protein changes were associated with corresponding changes in mRNA, indicating that the transcript level is a limited predictor of protein level. In addition, differential miRNA analyses showed that most miRNA changes in 231-BR were up- (36/45) as compared with down-regulations (9/45). Pathway analysis revealed that proteome changes were mostly related to cell signaling and cell cycle, metabolism and extracellular matrix remodeling. The major protein changes in 231-BR were confirmed by parallel reaction monitoring mass spectrometry and consisted in increases (by more than fivefold) in the matrix metalloproteinase-1, ephrin-B1, stomatin, myc target-1, and decreases (by more than 10-fold) in transglutaminase-2, the S100 calcium-binding protein A4, and L-plastin. The clinicopathological significance of these major proteomic changes to predict the occurrence of brain metastases, and their potential value as therapeutic targets, warrants further investigation.

The neuronal membrane protein sortilin has been reported in a few cancer cell lines, but its expression and impact in human tumors is unclear. In this study, sortilin was analyzed by immunohistochemistry in a series of 318 clinically annotated breast cancers and 53 normal breast tissues. Sortilin was detected in epithelial cells, with increased levels in cancers, as compared to normal tissues (p = 0.0088). It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001). There was an association between sortilin expression and lymph node involvement (p = 0.0093), suggesting a relationship with metastatic potential. In cell culture, sortilin levels were higher in cancer cell lines compared to non-tumorigenic breast epithelial cells and siRNA knockdown of sortilin inhibited cancer cell adhesion, while proliferation and apoptosis were not affected. Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation. In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.

Proteogenomics, or the integration of proteomics with genomics and transcriptomics, is emerging as the next step towards a unified understanding of cellular functions. Looking globally and simultaneously at gene structure, RNA expression, protein synthesis and posttranslational modifications have become technically feasible and offer a new perspective to molecular processes. Recent publications have highlighted the value of proteogenomics in oncology for defining the molecular signature of human tumors, and translation to other areas of biomedicine and life sciences is anticipated. This minireview will discuss recent developments, challenges and perspectives in proteogenomics.

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

Nerve growth factor (NGF) and its precursor (proNGF) are primarily considered as regulators of neuronal function that induce their responses via the tyrosine kinase receptor TrkA and the pan-neurotrophin receptor p75NTR. It has been generally held that NGF exerts its effects primarily through TrkA, inducing a cascade of tyrosine kinase-initiated responses, while proNGF binds more strongly to p75NTR. When this latter entity interacts with a third receptor, sortilin, apoptotic responses are induced in contrast to the survival/differentiation associated with the other two. Recent studies have outlined portions of the downstream phosphoproteome of TrkA in the neuronal PC12 cells and have clarified the contribution of individual docking sites in the TrkA endodomain. The patterns observed showed a similarity with the profile induced by the epidermal growth factor receptor, which is extensively associated with oncogenesis. Indeed, as with other neurotrophic factors, the distribution of TrkA and p75NTR is not limited to neuronal tissue, thus providing an array of targets outside the nervous systems. One such source is breast cancer cells, in which NGF and proNGF stimulate breast cancer cell survival/growth and enhance cell invasion, respectively. This latter activity is exerted via TrkA (as opposed to p75NTR) in conjunction with sortilin. Another tissue overexpressing proNGF is prostate cancer and here the ability of cancer cells to induce neuritogenesis has been implicated in cancer progression. These studies show that the non-neuronal functions of proNGF/NGF are likely integrated with their neuronal activities and point to the clinical utility of these growth factors and their receptors as biomarkers and therapeutic targets for metastasis and cancer pain.

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, tB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves.